title: Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity.
abstract: All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose.
authors.affiliation: From the Department of Chemistry.
authors.forename: Xinran
authors.initials: X
authors.lastname: Liu
authors.affiliation: From the Department of Chemistry.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Yang
authors.affiliation: the Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Cheri A
authors.initials: CA
authors.lastname: Lee
authors.affiliation: the Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Ibrahim M
authors.initials: IM
authors.lastname: Moustafa
authors.affiliation: the Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Smidansky
authors.affiliation: From the Department of Chemistry.
authors.forename: David
authors.initials: D
authors.lastname: Lum
authors.affiliation: the Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Jamie J
authors.initials: JJ
authors.lastname: Arnold
authors.affiliation: the Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
authors.affiliation: From the Department of Chemistry. Electronic address: ddb12@psu.edu.
authors.forename: David D
authors.initials: DD
authors.lastname: Boehr
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D006801:Humans; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D023321:Poliovirus Vaccines / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012254:Ribavirin / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014774:Virulence / Q000235:genetics
chemical_list: D023321:Poliovirus Vaccines; D011994:Recombinant Proteins; D014764:Viral Proteins; D012254:Ribavirin; D012324:RNA-Dependent RNA Polymerase
keywords: Enzyme Kinetics; NMR; Positive-strand RNA Viruses; Protein Dynamics; Viral Polymerase